Paclitaxel (available on the market as Taxol® Injection) is the prototype of chemotherapeutic taxanes, which binds to β-tubulin, promotes the assembly of this protein into microtubules and stabilizes them, eventually causing cell death. Paclitaxel poliglumex (PPX, chemical name N-(L-pyroglutamyl)-[poly(L-glutamyl)]-L-glutamic acid partially γ-esterified with (2′R,3′S)-3′-benzoylamido-1′-[[4,10β-bis(acetoxy)-2α-(benzoyloxy)-1,7β-dihydroxy-9-oxo-5,20-epoxytax-11-en-13α-yl]oxy]-1′-oxo-3′-phenylpropan-2′-ol), hereinafter referred to as PPX, is the ester conjugate of α-poly-L-glutamic acid (PGA) and paclitaxel wherein the latter is covalently bound to PGA through the 2′-hydroxy position.
PPX is currently under study for use in non-small cell lung cancer (NSCLC) and ovarian cancer. Paclitaxel is released after uptake and proteolytic/hydrolytic degradation of the conjugate in a tumor tissue.
The molecular formula and molecular weight of PPX are distributions because the polymerization degree of α-poly-L-glutamic acid and the number of conjugation sites with paclitaxel vary according to the manufacturing process. The average molecular mass of PPX is approximately 40,000 Daltons, determined by non-aqueous gel permeation chromatography with detection by multi angle laser light scattering. Approximately 35% by weight paclitaxel is present in the bound form in the conjugate, which amounts to about one paclitaxel ester linkage per 11 monomer units. Methods for the synthesis of PPX are disclosed in PCT publication WO97/33552.
Among manufacturing tests to be performed on PPX, both as active ingredient and as finished pharmaceutical product, assays for the quantification of conjugated paclitaxel and related taxane-based impurities are prescribed. However, the latter assay cannot be carried out on PPX as such and deesterification is required to liberate paclitaxel. Tetrahedron Letters, (1995), 36(12), 2001-2004 discloses a procedure for the hydrolytic cleavage of various esters of paclitaxel using basic hydrogen peroxide/tetrahydrofuran mixtures, wherein the 2′-acetate of peracetylated paclitaxel is hydrolyzed faster than the 10-acetyl position. When this method is applied to paclitaxel, the reported product is 10-deacetylpaclitaxel (10-DAT). A known procedure initially employed for the quantification of conjugated paclitaxel and related taxane impurities in PPX comprises exhaustive aqueous hydrolysis of PPX with sodium bicarbonate/peroxide with concomitant extraction of paclitaxel and related taxane impurities in CHCl3 and quantification by HPLC. The addition of CHCl3 as a second phase allows for extraction of the liberated taxanes immediately after hydrolysis, preventing most of the 10-DAT formation. The hydrolysis reaction, however, causes significant degradation of paclitaxel, principally to 10-deacetylpaclitaxel (10-DAT) and 20% levels of 10-DAT were common using this method, as the biphasic system did not completely suppress hydrolysis at the 10 position. As a result, the peaks of both paclitaxel and 10-DAT are quantified together and reported as paclitaxel+10-DAT. To be able to correct the amount of paclitaxel+10-DAT obtained from the hydrolysis method to total paclitaxel only, a second method using NMR quantification needs to be performed to quantify the amount of indigenous conjugated 10-DAT. Thus, the formation of degradation products and the change of the conjugated taxane profile is a clear drawback of this procedure. Moreover, the chromatographic conditions are not specific for the degradation compound.
There is therefore the need for an improved method for the quantification of PGA-conjugated paclitaxel and other PGA-conjugated taxanes.